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Fig. 4. Association of Smad1/Tcf4/ß-catenin molecular complexes with oligo-duplexes encoding either TBE-A or SBE-A. EMSA showing inhibition of migration generated by incubating nuclear extracts isolated from wild-type or TgAlk3^QD (QD) kidney tissue with ³²P-labeled oligo-duplexes corresponding to wild-type and mutant consensus binding regions within TBE-A and SBE-A (broken arrow). Unbroken arrows mark enhanced inhibition of migration associated with addition of specific antibodies. Free probe (FP) is observed at the lower end of each autoradiogram. (A) Left panel: migration of ³²P-labeled oligo-duplex, TBE-A, was retarded by addition of nuclear extract (broken arrow). The amount of oligo-duplex bound by extract from QD was greater than that from wild type. Addition of specific antibody caused a further migratory retardation (unbroken arrow). Association of Tcf4 was detected in both wild type and QD but at much higher levels in QD. Association of ß-catenin was detected at low levels in wild type and at much higher levels in QD. Association of Smad1 with TBE-A sequences was observed only in QD. The mobility of the supershifted band was greater in QD lysates compared with wild-type lysates. Right panel: specificity of TBE-A is demonstrated by failure to detect any specific retardation using a mutant version of TBE-A. (B) The specificity of DNA-protein interactions (broken arrow) is shown by progressive diminution of binding in the presence of tenfold and 100-fold excess of unlabeled oligo-duplex. (C) The specificity of antibody-mediated supershifts (unbroken arrow) is shown by the absence of a supershift in the presence of non-immune mouse or rat antisera. (D) Left panel: migration of ³²P-labeled oligo-duplex, SBE-A, was retarded by addition of nuclear extract (broken arrow). The amount of oligo-duplex bound by extract from QD was greater than that from wild type. Addition of specific antibody caused a further migratory retardation (unbroken arrow). Association of Smad1 with SBE-A was detected in both wild type and QD with greater amounts detected in QD. Association of ß-catenin was detected in wild type and at much higher levels in QD. Association of Tcf4 was detected only in QD. The mobility of the supershifted band was greater in QD lysates compared with wild-type lysates. Right panel: specificity of SBE-A is demonstrated by failure to detect any specific retardation using a mutant version of SBE-A. (E) The specificity of DNA-protein interactions (broken arrow) is shown by progressive diminution of binding in the presence of tenfold and 100-fold excess of unlabeled oligo-duplex. (F) The specificity of antibody-mediated supershifts (unbroken arrow) is shown by the absence of a supershift in the presence of non-immune mouse or rat antisera.

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